Method for radiolabeling antibodies with yttrium-90

ABSTRACT

Methods and kits for radiolabeling proteins, peptides and ligands with radiolytic isotopes, particularly yttrium-90, are disclosed, whereby sufficient purity, specific activity and binding affinity are achieved such that the radiolabeled protein may be directly administered to a patient without further column purification. Such kits and methods will be particularly useful in bringing radioimmunotherapy to the hospital and outpatient setting for the treatment of cancer.

This application claims the benefit of priority of and is a continuationof U.S. application Ser. No. 11/181,811, filed Jul. 15, 2005, now U.S.Pat. No. 7,229,620, which is a continuation of U.S. application Ser. No.09/628,186, filed Jul. 28, 2000, now U.S. Pat. No. 6,994,840, which is adivisional of U.S. application Ser. No. 09/259,338, filed Mar. 1, 1999,now abandoned.

FIELD OF THE INVENTION

The present invention relates to kits and methods for radiolabelingproteins, peptides and ligands with therapeutic radioisotopes such thatthese radiolabeled agents may be administered directly to patientswithout the need for additional purification. Such kits and methods areparticularly applicable to labeling proteins and peptides withyttrium-90 (⁹⁰Y). By optimizing the radiolabeling protocol such that nofurther purification of the radiolabeled protein is required, thepresent invention has satisfied a long-felt need in the art by solvingthe persistent problem of how to provide yttrium-labeled drugs in auser-friendly format such that these drugs may be easily prepared andadministered in the hospital or outpatient setting.

TECHNOLOGY BACKGROUND

All publications and patent applications herein are incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated to beincorporated by reference.

Radiolabeled proteins, particularly antibodies, have been undergoingevaluation for many years as potential diagnostic and therapeuticreagents. Such reagents are thought to be particularly useful as cancertherapeutics, now that researchers are beginning to identifytumor-specific antigens and cognate ligands or antibodies which bind tosuch antigens. By administering a radiolabeled ligand or antibody whichhas binding specificity for a tumor-specific antigen, coupled to aradioisotope that has a short range, high energy and abundant particleemission, one has the potential to deliver a lethal dose of radiationdirectly to the tumor cell.

Depending on the particle range of the particular isotope, labels may bechosen based on their suitability for targeting a particular type ofcell. For instance, gamma emitters are generally used for diagnosticpurposes, i.e., visualizing tumors, but are generally ineffective askilling agents. In contrast, alpha and beta emitters may be used toeffect cell killing. Alpha emitters may be particularly useful forblood-born diseases or vascular tumors where they can achieve goodpenetration; although one particle emission in some cases may be enoughto effect cell killing, typically alpha emitter must be located right atthe cell surface. In contrast, beta emitters, i.e., ⁹⁰Y, areparticularly suitable for bulkier, more localized disease because theytypically have a longer emission range.

Yttrium-90-labeled antibodies and peptides in particular have shownencouraging results in clinical therapy protocols (Thomas et al. 1995.Gamma-interferon administration after ⁹⁰Y radiolabeled antibody therapy:survival and hematopoietic toxicity studies. Int. J. Radiat. Oncol.Biol. Phys. 31: 529-534; DeNardo et al. 1995. Yttrium-90/Indium-111 DOTApeptide chimeric L6: pharmacokinetics, dosimetry and initial therapeuticstudies in patients with breast cancer. J. Nucl. Med. 36: 97P). Suchconjugates are usually made by coupling a bifunctional chelator to theprotein or antibody, then conjugating the radiolabel to the proteinconstruct via the bifunctional chelator. For instance, copendingapplication Ser. Nos. 08/475,813, 08/475,815 and 08/478,967, hereinincorporated by reference, describe radiolabeled therapeutic antibodiesfor the targeting and destruction of B cell lymphomas and tumor cells.Particularly disclosed is the Y2B8 conjugate, which comprises ananti-human CD20 murine monoclonal antibody, 2B8, attached to ⁹⁰Y via abifunctional chelator, MX-DTPA.

Patents relating to chelators and chelator conjugates are known in theart. For instance, U.S. Pat. No. 4,831,175 of Gansow is directed topolysubstituted diethylenetriaminepentaacetic acid chelators and proteinconjugates containing the same, and methods for their preparation. U.S.Pat. Nos. 5,099,069, 5,246,692, 5,286,850, and 5,124,471 of Gansow alsorelate to polysubstituted DTPA chelators. As described in Kozak et al.,several DTPA chelating agents, including MX-DTPA, have been shown to besuitable for yttrium-monoclonal antibody radioimmunotherapy (1989.Nature of the bifunctional chelating agent used for radioimmunotherapywith yttrium-90 monoclonal antibodies: Critical factors in determiningin vivo survival and organ toxicity. Cancer Res. 49: 2639-2644). Thesereferences are incorporated herein in their entirety.

Yttrium-90 is particularly suited for radioimmunotherapy and radioligandtherapy for several reasons. The 64 hour half-life of ⁹⁰Y is long enoughto allow antibody accumulation by the tumor and, unlike e.g., ¹³¹I, itis a pure beta emitter of high energy (E max 2.27 MeV) with noaccompanying gamma irradiation in its decay. It's particle emissionrange is 100 to 1000 cell diameters, which is a sufficiently minimalamount of penetrating radiation that outpatient administration would bepossible. Furthermore, internalization of labeled antibodies is notrequired for cell killing, and the local emission of ionizing radiationshould be lethal for adjacent tumor cells which might lack the targetantigen.

However, despite the recognized utility of yttrium-labeled antibodiesand the encouraging clinical results with some yttrium-labeledtherapeutics, many patients are deprived of the benefit thesetherapeutics might offer because of the inherent difficulties inconducting both the radiolabeling and administration at a singlelocation. This significant problem is evident in the nearly completevoid of kits and products which enable on-site labeling of reagents withalpha and beta emitting radioisotopes, which might otherwise demonstratethe commercial applicability of such technology.

The problem with providing kits for radiolabeling and subsequentadministration of therapeutics labeled with destructive isotopes appearsto be the long-existing belief in the art that, before such therapeuticscould be administered to a patient, an extensive purification processwas required to remove unbound label so as not to expose the patient tofree radioisotope which might accumulate in the bone and othernon-target organs. Even those kits currently available for labelingantibodies with yttrium require a complicated purification step beforethe therapeutic is ready for administration.

For instance, Antisoma currently offers a kit for radiolabelingmonoclonal antibody HMFG1 (Theragyn®) with ⁹⁰Y for subsequentadministration to patients who have been diagnosed with ovarian cancer.An extended phase I-II study demonstrated that this treatment may beparticularly beneficial to patients as a follow-up to conventionalsurgery and chemotherapy (Hird et al. 1993. Adjuvant therapy of ovariancancer with radioactive monoclonal antibody. Br. J. Cancer 68: 403-406).Yet Antisoma's labeling method requires removal of unbound label bySephadex G50 gel filtration, which is a significant deterrent to theTheragyn® labeling kit achieving commercial success, as well as anobstacle for ensuring that this therapy is readily available for allovarian cancer patients for whom it might serve to benefit.

The fact that such reagents currently require purification beforeadministration has been and will continue to be a major deterrent intheir availability to all patients who could benefit from suchtechnology unless a simplified method is presented that allowsphysicians to quickly, efficiently and safely administer such reagents.For instance, a doctor in an outpatient setting does not have the timeor facilities to purify a reagent by HPLC or gel filtrationchromatography before administering the reagent to his patient. Thismeans that additional facilities must be available on site forconcurrent production of the reagent and immediate delivery to thedoctor, which drastically increases the cost of the therapy and in somecases might require a patient to travel a significant distance toreceive the therapy. Alternatively, the drug could be labeled off-site,which would require prior preparation and at least a short-term storageof the therapeutic. This not only has the effect of decreasing thestrength of the radioisotope through radioactive decay during storage,but also leads to significant radiolytic damage to the structuralintegrity of the protein by overexposure to the radioisotope.

For instance, many reports have discussed the radiolytic nature of ⁹⁰Yand similar radioisotopes (i.e., Salako et al. 1998. Effects ofradiolysis on yttrium-90-labeled Lym-1 antibody preparations. J. Nucl.Med. 39: 667-670; Chakrabarti et al. 1996. Prevention of radiolysis ofmonoclonal antibody during labeling. J. Nucl. Med. 37: 1384-1388). Asnoted in Chakrabarti et al., radionuclides such as ⁹⁰Y deliver a largeamount of radiation to the antibody during the labeling process as wellas during storage. Radiation has reportedly led to instances ofsignificant antibody damage, which can eliminate preferential targetingof tumor cells and expose non-target tissues to significant levels oftoxicity.

The mechanism for radiation damage has been attributed to the generationof free radicals (Pizzarello. 1975. Direct and indirect action. In:Pizzarello and Witcofski, eds. Basic Radiation Biology, 2^(nd) ed.Philadelphia: Lea & Febger, pp. 20-29). But as noted in Salako et al.,at an energy of 2.2 MeV, the beta particles emitted from ⁹⁰Y couldeasily break most chemical bonds including the disulfide bridges of anantibody, which have a bond strength of only 4.4 eV (Skoog. 1985.Principles of Instrumental Analysis, 3^(rd) edition. San Francisco:Saunders). Thus, the shorter the amount of time that the protein to belabeled is exposed to destructive radioisotopes such as ⁹⁰Y, the betterthe chances will be that the protein will maintain the structuralintegrity and binding specificity it requires to interact with thetarget antigen up until the time it is administered and reaches thetarget site.

The radiolytic nature of ⁹⁰Y has been known in the art for years andmany have tried to solve the problem ⁹⁰Y presents in the commercialapplication of these therapeutics. For instance, both Salako et al. andChakrabarti et al. evaluate the use of radioprotectants in ⁹⁰Y-labeledantibody preparations as a means to decrease damage to the antibody.Salako et al. in particular reported that human serum albumin enabledmaintenance of ⁹⁰Y-labeled antibody immunoreactivity for up to 72 hours.However, the specific activity exhibited by Salako's preparations wasrather low (less than 2 mCi/ml). Moreover, neither Salako norChakrabarti report any effort to forego the extensive purificationprocesses required after antibody labeling. Salako et al. labels for aperiod of 45 minutes to an hour, then purifies the antibody by molecularsieve chromatography, whereas Chakrabarti labels for nearly three hoursand purifies by gel filtration chromatography. Neither of these methodswill be instrumental in bringing ⁹⁰Y-labeled therapeutics to theout-patient setting.

Chinol and Hnatowich were able to achieve 90% radiochemical purity for⁹⁰Y-labeled proteins with specific activities ranging from 1-3 mCi/mgabsent post-labeling purification, using their own generator-produced⁹⁰Y (1987. Generator-produced yttrium-90 for radioimmunotherapy. J.Nucl. Med. 28(9): 1465-1470). However, the authors expressly discourageadministering preparations having less than 95% purity to patients, andsuggest that HPLC may be an important and “possibly essential” step.

Those who have recognized that HPLC and other types of purification mustbe eliminated in the outpatient and hospital setting have not succeededin developing a sufficient labeling protocol for ⁹⁰Y such that a highlevel of label incorporation is achieved and an acceptable level ofantibody stability is maintained. If a high level of radioincorporationis not consistently achieved, the patient could be exposed tounacceptable levels of free non-bound radiolabel if this label is notpurified away from the reagent. Moreover, again, if antibody structuralintegrity is damaged such that the antibody loses target specificity,such reagents will not bind specifically to their cognate ligands.

Mather and colleagues set out with the purpose of labelingtumor-specific antibodies with ⁹⁰Y in a manner such that post-labelingpurification could be avoided (1989. Labeling monoclonal antibodies withyttrium-90. Eur. J. Nucl. Med. 15: 307-312). However, Mather found thathigh labeling efficiencies (over 95%) could only be achieved at modestspecific activities (1 mCi/mg). Moreover, Mather et al. reports thattheir antibody preparations showed signs of breakdown (due toradiolysis) after only a few hours. This may be because Mather et al.,as do many others in the field, conducted their labeling reaction over aperiod of one hour.

For example, there have been methods proposed for labeling proteinreagents with less destructive labels such as ¹¹¹In which foregoadditional purification steps. Richardson et al. propose such aprocedure for labeling antibodies with ¹¹¹In with the goal offacilitating a kit format for diagnostic use (Richardson et al. 1987.Optimization and batch production of DTPA-labeled antibody kits forroutine use in ¹¹¹In immunoscintography. Nucl. Med. Comm. 8: 347-356).However, the labeling method proposed in Richardson et al. is conductedover a period of one hour, which might be feasible with ¹¹¹In which isnot very radiolytic, but does not appear to be amenable to ⁹⁰Y labelingapplications as evidenced by the difficulties reported in Mather et al.

This brings us to the surprising and unexpected advantages of thepresent invention, which provides invaluable insight into the process ofradiolabeling proteins with ⁹⁰Y which has not been yet been recognizedby others in the art. Surprisingly, the present inventors have foundthat the processes of HPLC or other purification steps that others havelong thought to be necessary to achieve pure reagent, and the lengthyincubation times which others have adopted in an effort to increase thespecific activity of their reagents, are actually detrimental to theprocess of preparing ⁹⁰Y-labeled reagents. Such time-inclusive processesserve only to increase the damage to the protein due to radiolysis,leading to less specificity or binding, loss of radioisotope from thetargeting agent and an increased rate of protein degradation by the timethe radiolabeled protein is ready for injection. Surprisingly, thepresent inventors have found that efficient labeling with ⁹⁰Y (>95%incorporation and at least 15 mCi/mg specific activity) can beaccomplished in as little as two to five minutes, and in fact suchlabeling loses its efficiency as reaction times are increased beyondeven eight minutes.

The fact that labeling with ⁹⁰Y may now be achieved by the methods ofthe present invention in as little as one-two minutes or even as quicklyas 30 seconds will completely dissolve the current skepticism in thefield toward the applicability of yttrium radiolabeling kits in hospitaland outpatient settings. The kits of the present invention will thereforfinally satisfy the long felt need that has perhaps been recognized bymany cancer patients and doctors alike with regard to the commercialapplicability and accessability of protein-based, radiolabeled cancertherapeutics.

SUMMARY OF THE INVENTION

The present invention concerns methods and kits for radiolabeling achelator-conjugated protein or peptide with a therapeutic radioisotopefor administration to a patient. The methods of the present inventionessentially comprise (i) mixing the chelator-conjugated protein, ligandor peptide with a solution comprising the radioisotope or a saltthereof, and (ii) incubating the mixture for a sufficient amount of timeunder amiable conditions such that a radiolabeled protein or peptidehaving sufficient purity, i.e., level of radioincorporation, specificactivity and binding specificity is achieved such that the radiolabeledantibody may be administered directly to the patient without furtherpurification.

The kits of the present invention essentially comprise (i) a vialcontaining chelator-conjugated protein or peptide, (ii) a vialcontaining formulation buffer for stabilizing and administering theradiolabeled antibody to a patient, and (iii) instructions forperforming the radiolabeling procedure, such that when thechelator-conjugated protein or peptide is exposed to the radioisotope ora salt thereof for a sufficient amount of time under amiable conditionsas recommended in said instructions, a radiolabeled protein or peptidehaving sufficient purity, specific activity and binding specificity isachieved such that the radiolabeled antibody may be diluted to anappropriate concentration in said formulation buffer and administereddirectly to the patient without further purification. It be noted thatthe sterile vial format of the disclosed kits also forego the need forsterility or endotoxin testing, thereby further simplifying the kit andmaking it more user-friendly.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A) SB cells were washed and resuspended to 90×10⁶ cells/mL withdilution buffer (1×PBS, pH 7.4 containing 1% (w/v) bovine serum albumin.Increasing concentrations of cells were incubated for 3 h with 2 ng/mLY2B8 prepared using 2B8-MX-DTPA lot #0165A. B) Double-inverse plot ofcell concentration vs. bound radioactivity/total radioactivity (B/AT).Immunoreactivity was calculated as 1/y-intercept×100. Immunoreactivityand correlation coefficient (R) values were 72.2% and 0.999,respectively.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described.

The present invention includes a method for radiolabeling achelator-conjugated protein, ligand or peptide with a therapeuticradioisotope for administration to a patient comprising (i) mixing thechelator-conjugated protein or peptide with a solution comprising theradioisotope or a salt thereof, and (ii) incubating the mixture for asufficient amount of time under amiable conditions such that aradiolabeled protein or peptide having sufficient purity, i.e., level ofradioincorporation, specific activity and binding specificity isachieved such that the radiolabeled antibody may be administereddirectly to the patient without further purification. “Furtherpurification” includes HPLC, gel filtration, other types of columnchromatography and any other separation technique which is employed withthe purpose of removing free or bound unconjugated radiolabel.

The methods of the present invention are particularly applicable totherapeutic radioisotopes which are typically radiolytic and thereforpotentially dangerous to the structural integrity of the protein. Suchtherapeutic radioisotopes are generally selected from the groupconsisting of alpha and beta emitters. Preferred therapeuticradionuclides include ²⁰³Pb, ²¹²Pb ²¹²Bi ¹⁰⁹Pd ⁶⁴Cu, ⁶⁷Cu, ⁹⁰Y, ⁷⁷Br,²¹¹At, ⁹⁷Ru, ¹⁰⁵Rh, ¹⁹⁵Au and ¹⁹⁹Ag or ¹⁷⁷Lu. Other radionuclides whichhave therapeutic utility are described in U.S. Pat. No. 5,541,287,herein incorporated by reference. Particularly preferred radionuclidesare strong beta radiation emitters which may cause intramoleculardecomposition, such as ⁹⁰Y, ⁶⁷Cu, ¹³¹I, ¹⁸⁶Re and ¹⁸⁸Re. Although a“therapeutic” radioisotope generally refers to radioisotopes such asbeta and gamma emitters which have a cytotoxic affect, to the extentthat such radioisotopes may also be used for diagnostic purposes, suchpurposes do not remove these isotopes from the scope of the presentinvention because it is the radiolytic nature of these isotopes whichrenders them suitable for the disclosed methods and kits.

The methods of the present invention may be used to label proteins,ligands or peptides, particularly protein ligands where structuralintegrity must be maintained for target specificity. Preferred proteinsare antibodies or antibody fragments, such as Fab, (Fab)₂, and Fvfragments, which recognize tumor specific or tumor-associated antigens.Preferred peptides include somatostatin, vasointestinal peptide (VIP),substance P and others which bind to cellular receptors. Such peptidesand chelator-conjugated derivatives of such peptides are disclosed inU.S. Pat. No. 5,830,431, herein incorporated by reference.

A “sufficient incubation time” as referenced in the methods of theinvention is the acceptable time range during which sufficientradioincorporation and radiochemical purity are achieved such that thereagent may be administered directly to a patient without the need forfurther purification. Sufficient radioincorporation and purity isgenerally recognized in the art to be at least 95% but may varydepending on the toxicity of the label. It should also be apparent tothose of skill in the art that the extent of radioincorporationconsidered to be sufficient is also a function of the desired level ofefficacy. For ⁹⁰Y, and particularly the ⁹⁰Y-labeled antibodies of thepresent invention, such sufficient time may be generally less than abouteight minutes, and more preferably between about two to about fiveminutes, given an amenable molar ratio of chelator to protein in thechelator-conjugated protein to be labeled.

It should be apparent to those of skill in the art that the optimum timerequired for labeling a specific protein may vary depending on theprotein, the particular radiolabel and the particular conjugateemployed. An underlying factor in the optimization of the time allottedfor radiolabeling is the chelator to protein ratio of the reagent whichis to be labeled. For instance, the chelator to protein ratio must behigh enough to achieve a therapeutically useful level of incorporation,i.e., 95%, but must also not be too high such that the structuralintegrity or immunoreactivity of the protein is compromised. Thisrequires a certain balancing process that in some cases may lead to alower level of conjugated chelator and longer labeling time.

For instance, the present inventors have discovered that labeling with⁹⁰Y to the desirable level of purity may be accomplished in under fiveminutes using MX-DTPA as a chelator and only about a 1½ to 1 molar ratioof chelator to antibody. Although the chelator to antibody ratio couldactually be increased, this was not necessary because a desirable levelof radioincorporation and specific activity was achieved after a shortlabeling period. Given this discovery, parameters such as chelator toprotein concentrations could be readily determined by empirical means bythose of skill in the art for other proteins and peptides, depending onthe therapeutic label of choice, the choice of chelator the number ofsites available for chelator attachment, susceptibility of the proteinto radiolysis, desired level of efficacy, etc.

Any bifunctional chelator may be used in the method of the presentinvention so long as it is capable of binding to both the protein andradioisotope of interest. Preferred chelators may be selected from thegroup consisting of MX-DTPA, phenyl-DTPA, benzyl-DTPA, CHX-DTPA, DOTAand derivatives thereof. A particularly preferred chelator is MX-DTPA.

“Amiable conditions” as referenced in the present methods includeacceptable temperature, pH and buffer conditions. It should be apparentto those of skill in the art that reaction conditions should not bechosen that are inhibitory or otherwise not conducive to the labelingreaction. Lewis et al. discusses reaction conditions to be consideredwhen radiolabeling immunoconjugates, and is herein incorporated byreference (1994. A facile, water-soluble method for modification ofproteins with DOTA. Use of elevated temperature and optimized pH toachieve high specific activity and high chelate stability inradiolabeled immunoconjugates. Bioconjugate Chem. 5: 565-576).

An acceptable temperature for the reaction may vary depending on theprotein to be labeled, but in general ranges from about 25° C. to about43° C. Lewis et al. have found that increasing the temperature of theradiolabeling reaction from 25° C. to 43° C. increased both theefficiency of radiometal incorporation and the kinetic stability of theDOTA radioconjugates examined.

An acceptable pH may vary considerably depending on the radiolabel to beused. The recommended pH for labeling with different radionuclides isgenerally known in the art and may be chosen accordingly in view of theradioisotope. For instance, for ⁹⁰Y, an acceptable pH may range fromabout 3 to about 6, but is more preferably about 4.2.

Acceptable buffers will also vary depending on the particularradiolabel. For instance, Lewis et al. and others have found that thepresence of citrate inhibits labeling reactions with ⁹⁰Y. Thus, citratebuffer would not be appropriate if the ⁹⁰Y were the chosen radiolabel.When labeling with ⁹⁰Y, the preferred buffer is an acetate buffer, andmore particularly a sodium acetate buffer at a concentration of betweenabout 10 and about 1000 mM.

If it does not inhibit or otherwise adversely affect the labelingreaction, it may also be possible for a benign (non-adverse)radioprotectant to be included in the reaction buffer. According toChakrabarti, ascorbic acid is one such radioprotectant which does notinterfere with the labeling process. Caution should be exercised,however, when employing human serum albumin in the labeling reaction dueto the presence of metals which would interfere with the labelingprocess. HSA (as well as other radioprotectants) may need to be treatedto lower metal content so that acceptable incorporation is obtained.

Because the present invention concerns radiolabeling proteins withparticularly radiolytic isotopes, there may be a certain balance betweenbinding specificity and specific activity that the skilled artisan mayencounter when practicing the methods of the present invention. Forinstance, when specific activity is very high (i.e., suitably over 5mCi/mg, preferably over 10 mCi/mg and more preferably over 15 mCi/mg), aprotein construct having the desired binding specificity will have asignificant killing capability at the region of the tumor. However, theportion of proteins in the population as a whole which retain theirimmunoreactivity may be lower than a population having a lower specificactivity due to radiolysis of the radiolabel. Depending on the desiredlevel of specific activity, the skilled artisan may choose to compromisea certain level of immunoreactivity.

For instance, the present inventors have found that, with ⁹⁰Y, when anantibody is labeled to a specific activity of about 15 mCi/mg, thebinding specificity or immunoreactivity of the protein is generally atleast about 70%. This of course may vary depending on the sensitivity ofthe antibody and the radiolytic nature of the radioisotope employed, andmay be manipulated by the skilled artisan if a higher level ofimmunoreactivity or specific activity is desired. The present inventorshave achieved specific activities with ⁹⁰Y of up to about 20 mCi/mg.Binding specificities of at least 50% are desirable for therapeuticapplications.

Copending application Ser. Nos. 09/259,337 and 09/259,347, co-owned andsubmitted concurrently herewith, disclose binding assays which may beused to assess the percent binding affinity and immunoreactivity ofconjugates after labeling if desirable. It should be stressed that,although no further purification is required after the labeling methodsof the present invention, a TLC-based assay to verify the level ofradioincorporation should always be performed so as not to jeopardizethe health of the patient. Such an assay can be performed in about 3-4minutes, and should not significantly affect the stability or efficacyof the radiotherapeutic.

The present invention also includes kits for radiolabeling achelator-conjugated protein or peptide with a therapeutic radioisotopefor administration to a patient comprising (i) a vial containingchelator-conjugated protein or peptide, (ii) a vial containingformulation buffer for stabilizing and administering the radiolabeledantibody to a patient, and (iii) instructions for performing theradiolabeling procedure, such that when the chelator-conjugated proteinor peptide is exposed to the radioisotope or a salt thereof for asufficient amount of time under amiable conditions as recommended insaid instructions and described further above, a radiolabeled protein orpeptide having sufficient purity, specific activity and bindingspecificity is achieved such that the radiolabeled antibody may bediluted to an appropriate concentration in said formulation buffer andadministered directly to the patient without further purification. Saidchelator-conjugated protein or peptide may be supplied in lyophilizedform.

It should be understood that the kits of the present invent are designedto accomplish the methods described herein and may therefor be used forthat purpose. Accordingly, it should be apparent to those concerned whenreading the invention that the kit instructions will be based on themethods described above, and that the considerations addressed abovehave the same relevance and meaning when considered in view of the kitembodiment. Additionally, it should be apparent upon reading thedisclosure as a whole that alternative kit embodiments are encompassedin the present invention which may contain components such as an acetatebuffer for adjusting the pH of the radioisotope or of the protein asdescribed above.

A particularly advantageous component of the kit is the formulationbuffer for stabilizing against the effects of radiolysis andadministering the radiolabeled conjugated antibody to a patient. Theformulation buffer is a pharmaceutically acceptable carrier which servesas both a diluent for the labeled antibody and an administration buffer.Although any pharmaceutically acceptable diluent may be used foradministering therapeutic or diagnostic antibodies to patient, theformulation buffer of the present invention is particularly suited foradministering radiolabeled antibodies.

For instance, the formulation buffer of the present invention comprisesa radioprotectant such as human serum albumin (HSA) or ascorbate, whichminimize radiolysis due to yttrium and other strong radionuclides. Otherradioprotectants are known in the art and could also be used in theformulation buffer of the present invention, i.e., free radicalscavengers (phenol, sulfites, glutathione, cysteine, gentisic acid,nicotinic acid, ascorbyl palmitate, HOP(:O)H₂, glycerol, sodiumformaldehyde sulfoxylate, Na₂S₂O₅, Na₂S₂O₃, and SO₂, etc.).

The formulation buffer of the present invention also comprises excessunconjugated chelator. The purpose for including unconjugated chelatoris that this chelator serves to scavenge any non-protein boundradiolabel in the patient, and causes excretion of the radiolabelthereby reducing uptake of “bone-seeking” isotopes, i.e., ⁹⁰Y, by thebones of the patient. For instance, when the antibody of the kit isconjugated to a DTPA chelator, excess DTPA or any other chelator may beincluded in the formulation buffer. The formation buffer is alsopreferably supplied in a volume such that the entire contents aretransferred to the reaction vial. As discussed above, this results inincreased ease of use and reproducibility because exact volumes do nothave to be measured and transferred.

A preferred formulation buffer comprises phosphate buffered orphysiological saline, human serum albumin and DTPA. The human serumalbumin is preferably at a concentration of between about 5 to 25%(w/v), and more preferably at a concentration of about 7.5% (w/v). Theconcentration of DTPA is preferably about 1 mM. Ascorbate may be used asan alternative to human serum albumin, and is typically used at aconcentration of about 1 to 100 mg/mm. Although a wider range ofconcentrations may be used without compromising patient safety.

The kit may be supplied in other alternative embodiments depending onthe preferences of the purchaser. For instance, the kit may furthercomprise a sterile reaction vial in which the labeling reaction anddilution into formulation buffer may both be performed. Furtherembodiments are envisioned whereby the buffer for adjusting the pH ofthe radiolabel is supplied in the actual reaction vial to cut, down onwaste and increase ease-of-use. Also, envisioned is a kit where isotopeis provided in a “buffered” form allowing for direct addition of theantibody/peptide conjugate to the isotope vial which serves as areaction vial. Also, the conjugate could be provided in a “buffered”form allowing direct addition of isotope. Thus, included in theinvention are kits which further comprise a vial of radioisotope,although it may be more feasible to order the labeling kit in advanceand order the radioisotope separately at a later time just beforeadministration. Also envisioned are kits which comprise a vial ofsecondary protein or peptide to serve as either a control in assessingthe binding affinity of the radiolabeled product, or in some cases to beemployed in a combined therapeutic regimen with the radiolabeled proteinor peptide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A ⁹⁰Y-labeled murine monoclonal anti-CD20 antibody (Y2B8) is currentlybeing evaluated in clinical trials for the treatment of relapsed B-celllymphoma. The 2B8 antibody is a murine antibody which recognizes humanCD20. The chimeric version of this antibody (Rituxan®) has recentlyreceived FDA approval for the treatment of non-Hodgkin's lymphoma. U.S.application Ser. No. 08/475,813, herein incorporated by reference,discloses sequential administration of Rituxan® with yttrium-labeledmurine monoclonal antibody in a combined therapeutic regimen, whereinadministration of the yttrium-labeled anti-CD20 antibody followingadministration of Rituxan® is sufficient to (a) clear any remainingperipheral blood B cells not cleared by the chimeric anti-CD20 antibody;(b) begin B cell depletion from lymph nodes; or (c) begin B celldepletion from other tissues.

Thus, given the proven efficacy of an anti-CD20 antibody in thetreatment of non-Hodgkin's lymphoma, and the known sensitivity oflymphocytes to radioactivity, it would be highly advantageous for suchtherapeutic antibodies to become commercially available in kit formwhereby they may be readily modified with a radiolabel and administereddirectly to the patient in the clinical setting.

A radiolabeling kit for the 2B8 antibody is preferably comprised of fourcomponents: 1.) 2B8-MX-DTPA in low-metal normal saline at 2 mg/mL, 2.)50 mM sodium acetate used to adjust radioisotope solution to appropriatelabeling pH, 3.) formulation buffer (1×PBS, pH 7.4 containing 7.5% humanserum albumin and 1 mM DTPA), and optionally, 4.) an empty 10 mL glassvial (reaction vial) (a “10 mL” reaction vial actually holds 10 mLcomfortably, and is technically somewhat larger than “10 mL”). Allcomponents are tested to be sterile and pyrogen-free.

This section summarizes the validation of this radiolabeling kit whichis simple and easy to use and which yields radiolabeled antibodies with≧95% radioincorporation and acceptable retention of binding toantigen-positive cells. An evaluation of experimental parametersaffecting binding and radioincorporation was also conducted.

Example 1 Radiolabeling Kit and Method for Labeling 2B8 with ⁹⁰Y

A. Reagents in Radiolabeling Kit

-   1. 2B8-MX-DTPA, IDEC; Lot#082395RM2-   2. 50 mM Sodium Acetate, low-metal, IDEC; Lot#082395RM3-   3. Formulation Buffer (1×PBS, pH 7.4 containing 7.5% (w/v) human    serum albumin and 1 mM DTPA), IDEC, Lot#082395RM1-   4. Reaction vial, 10 mL, IDEC    B. Materials and Equipment-   1. Biodex Tec-Control Radioincorporation Kit, Cat.#151-770-   2. Gloves: powder-free-   3. Sterile polypropylene syringes-   4. Sterile syringe needles-   5. Small tubes with closure; 1.5 ml    C. Methods

1. Preparation of Y2B8 Using Radiolabeling Kit

Kit reagents were prepared and filled into glass septum vials. Type Iborosilicate vials (2 or 10 mL) were rinsed with sterile water forinjection (WFI) and autoclaved before filling. Butyl rubber septa wererinsed with sterile WFI and autoclaved before use. Reagents weremanually filled and crimped in a Class 100 room and tested forpyrogenicity and sterility using USP methods.

Additional Reagents:

-   1. Yttrium-[90]: chloride salt, carrier-free, in HCl.    Precautions:    1. All steps should be performed using aseptic technique.    2. Radiolabeling kit components should be allowed to come to room    temperature before use.    Radiolabeling Protocol    1. The volume of ⁹⁰YCl₃ to add to the reaction vial was calculated    as follows:

a. The radioactivity concentration at the time of radiolabeling:

-   -   C₀=Radioactivity concentration at time of calibration (see        manufacturer's Certificate of Analysis).    -   Δt=Change in time (positive number is post calibration, negative        number is pre calibration).

${{Radioactivity}\mspace{14mu}{Concentration}\mspace{14mu}{at}\mspace{14mu}{time}\mspace{14mu}{of}\mspace{14mu}{labeling}} = \frac{C_{0}}{{\mathbb{e}}^{0.0108{({\Delta\; t})}}}$

b. The volume of ⁹⁰YCl₃ to add to the reaction vial:

$\frac{45\mspace{14mu}{mCi}}{\begin{matrix}{{Radioactivity}\mspace{14mu}{Concentration}} \\{\;{{time}\mspace{14mu}{of}\mspace{14mu}{labeling}}}\end{matrix}\mspace{11mu}} = {{Volume}\mspace{14mu}{added}\mspace{14mu}{to}\mspace{14mu}{reaction}\mspace{14mu}{vial}}$2. The volume of 50 mM sodium acetate to add to the reaction vial wascalculated as follows:

-   -   a. For ⁹⁰YCl₃ in 0.040 M HCL (Amersham):    -   Volume ⁹⁰YCl₃ (Step 1b)×(0.8)=volume of sodium acetate to add    -   b. For ⁹⁰YCl₃ in 0.050 M HCl(Nordion):    -   Volume ⁹⁰YCl₃ (Step 1b)×(1.0)=volume of sodium acetate to add        3. The septa of the reaction vial and the sodium acetate vial        were wiped with alcohol. Using a 1 cc syringe, the calculated        volume (Step 1a or 1b) of 50 mM sodium acetate (Step 2) was        transferred to the reaction vial. The vial was mixed by        inverting several times.        4. The septum of the ⁹⁰YCl₃ source vial was wiped with alcohol.        The vial with a needle fitted with sterile 0.2 μm filter. Using        a 1 cc sterile syringe, was vented the required volume (Step 1b)        of ⁹⁰YCl₃ was transferred to the reaction vial. The vial was        mixed by inverting several times.        5. The septum of the 2B8-MX-DTPA vial was wiped with alcohol.        Using a 3 cc sterile syringe, 1.5 mL of 2B8-MX-DTPA was        transferred to the reaction vial. The vial was mixed by        inverting several times.        6. The total volume of reaction mixture was calculated by adding        the amount of Y-90 chloride added (Step 4), plus the amount of        50 mM sodium acetate added (Step 3), plus the amount of        2B8-MX-DTPA added (Step 5).        7. The volume of Formulation Buffer to add to the Reaction Vial        to obtain a final volume of 10 mL was calculated by subtracting        the total reaction volume calculated in step 6 from 10.        8. The Formulation Buffer vial was wiped with alcohol and the        vial was vented. Due to the viscosity of the Formulation Buffer,        the reaction vial using a needle fitted with a 0.20 μm syringe        filter. Using a 10 cc sterile syringe fitted with an appropriate        gauge needle, the volume of Formulation Buffer calculated in        Step 7 was transferred to the reaction vial. The vent needle was        removed from the reaction vial and the vial was mixed by        inverting several times (Final Product). The vial was incubated        at least 5 minutes prior to doing the “Radioincorporation        Assay”. The color of the solution was amber and the reaction        vial was full thereby confirming that Formulation Buffer was        added.        9. The total radioactivity of the Final Product vial was        measured using the appropriate instrumentation set for        measurement of ⁹⁰Y.        10. The Final Product was immediately stored at 2°-8° C. until        required for patient administration.

2. Radioincorporation Assay

Percent radioincorporation was determined by instant thin-layerchromatography (ITLC) using the Biodex Tec-Control RadiochromatographicKit according to the following protocol:

Additional Materials and Equipment:

1. ⁹⁰Y-radiolabeled 2B8MX-DTPA

2. Tubes for counting radioactive TLC strips

3. Scissors

4. Sterile syringe, 1 cc

5. Sterile needles, 26G

6. Gamma counter or scintillation counter

7. Pipettor

Procedure:

1. The entire Biodex Operation Manual should be read first.

2. Each radiolabeled sample in triplicate was tested according to kitinstructions; one strip per vial was developed.

3. To spot the radiolabeled sample on the chromatography strip, apipettor was used to spot 1 μl on the origin line. Alternatively, onesmall drop dispensed from a 26G needle attached to a sterile 1 ccsyringe may be spotted. The antibody remains at the origin, andunincorporated ⁹⁰Y-DTPA moves with the solvent front.4. Each section was counted for activity using an appropriate counter,i.e., a scintillation counter for ⁹⁰Y, adjusting for background.5. The Biodex instructions for calculating the percentage ofradiolabeled antibody were followed.

3. Binding Assay

Additional Reagents

1. ⁹⁰Y2B8-MX-DTPA

2. Lyophilized cells—

The human cell lines SB (CD20-positive) and HSB (CD20-negative) wereobtained from American Type Culture Collection and cultured in T-flasksusing RPMI-1640 containing 10% fetal bovine serum supplemented with 2%glutamine. Cultures were maintained at 37° C. and 5% CO₂. Cells weretypically split 1:2 every other day and harvested at 0.5-2.5×10⁶cells/mL and viability's >80%. Cell concentrations were determined usinga hemacytometer and viability determined by trypan blue exclusion.

Cells were harvested at ambient temperature at a cell density of0.5-2×10⁶ cells/mL by centrifugation (1300 rpm in a Sorvall centrifuge)and washed twice with 1×HBSS. Pelleted cells were resuspended to 50×10⁶cells/mL in 1×HBSS containing 1% (w/v) bovine serum albumin (BSA) and10% (w/v/) mannitol (lyophilization buffer), 0.5 mL dispensed into 1.5mL polypropylene microfuge tubes with o-ring gaskets and stored at −70°C., and lyophilized overnight at 30-60 millitorr. Tubes of lyophilizedcells were stored desiccated at 2-8° C. and reconstituted in sterilewater for assays; tubes of cells lyophilized in microfuge tubes werestored with desiccant.

3. Sterile water for irrigation or sterile water for injection

4. Dilution buffer (1×PBS, pH 7.2-7.4 containing 1% Bovine Serum Albumin(BSA), and 0.02% Sodium Azide)

Procedure:

Radiolabeled antibody sample prep

-   1. The radiolabeled antibody stored at 2°-8° C. was obtained.-   2. A volume of 10 μL was withdrawn with a P20 and added to a 1.5 mL    microfuge tube containing 990 μL of Dilution buffer (1:100    dilution). The tip was rinsed and the tube was vortexed slightly.-   3. A 50 mL sterile polypropylene tube with h cap was obtained and 10    mL of Dilution buffer to the tube, using a 10 mL serological    pipette.-   4. A volume of 35 μL was withdrawn with a P200 from the 1:100    dilution tube and added to the conical tube containing 10 mL of    Dilution buffer. Mix thoroughly.    Lyophilized Cell Prep-   1. Three tubes of lyophilized SB Cells were obtained.-   2. A volume of 0.5 mL of SWFI was added to each tube, and the tubes    were vortexed until single cell suspensions were obtained.-   3. Three empty 1.5 mL microfuge tubes were obtained; to three of the    tubes, 0.5 mL of Dilution buffer was added, representing a control    with no cells. Assay Protocol-   1. A volume of 0.5 mL of the diluted ⁹⁰Y2B8-MX-DTPA was added to    each tube.-   2. The tubes were placed on end over mixer for 45 minutes, after    making sure caps are securely tightened.-   3. After 45 minutes incubation at ambient temperatures the cells    were pelleted by microcentrifugation for 5 minutes.-   4. A volume of 0.8 mL of the supernatant was transferred to    scintillation vials.-   5. Scintillation cocktail was added to each vial.-   6. The amount of radioactivity in each vial was determined using a    scintillation counter, adjusting for background.    D. Results

Reproducibility and ruggedness of the radiolabeling protocols for Y2B8was evaluated by performing several validation runs using different lotsof each radioisotope. Six validation lots each of Y2B8 were prepared byfive operators. These lots were designated as follows and performed atthe following facilities:

-   -   #1: IDEC Pharmaceuticals    -   #2: IDEC Pharmaceuticals    -   #3: IDEC Pharmaceuticals    -   #4: MD Anderson Health Center    -   #5: Mayo Clinic    -   #6: City of Hope        The results of testing on each validation lot are summarized in        Table 1.

TABLE 1 Release Assay Results for Y2B8 Validation Lot Number %Radioincorporation % Binding 1 99.5 78.6 2 99.3 87.0 3 99.4 85.9 4 99.281.8 5 99.2 79.6 6 96.3 80.8 Mean = 98.8 Mean = 82.3 Standard Deviation= 1.24 Standard Deviation = 3.4 % CV = 1.25% CV = 4.2%

For the six validation lots prepared, the percent binding obtained wasin the ranged from 78.6% to 87.0% with a mean of 82.3%.Radioincorporation values for Y2B8 averaged 98.8% (range of 96.3% to99.5%). Together, these results confirm the reproducibility andruggedness of the radiolabeling kit methods for preparation of Y2B8, andtogether indicate that Y2B8 prepared using this radiolabeling kit aresuitable for use in the clinical setting.

Example 2 Initial Evaluation of the Reaction Parameters pH and ReactionTime

Kinetic studies were initially performed to evaluate theradioincorporation and binding of the ⁹⁰Y-labeled antibody (Y2B8)following labeling reactions performed under varying conditions of pHand reaction time. For radiolabeling reactions in the range of pH 3.9 to4.7 at an incubation time of 5 min, radioincorporation was >96%with >80% retention of binding to CD20-positive cells (Table 2). Similarresults were obtained for incubation times of 3, 5, and 10 min for therange of pH 2.9 to 4.6 (Table 3).

TABLE 2 Y2B8 Radiolabeling Kinetics: Effect of pH on Radioincorporationand Binding to CD20-Positive Cells¹ Reaction pH Radioincorporation (%)Binding (%) 3.9 98.4 80.7 4.2 97.8 81.0 4.4 96.1 80.0 4.6 97.0 80.2 4.797.4 80.6

TABLE 3 Y2B8 Radiolabeling Kinetics: Effect of Incubation Time onRadioincorporation and Binding to CD20-Positive Cells¹ Incubation Time(min) Radioincorporation (%) Binding (%) pH 3.9: 3 97.0 82.0 5 98.9 82.110 99.2 82.3 pH 4.7: 3 97.2 82.5 5 96.7 81.8 10 97.6 81.5 ¹The labelingreaction results and parameter evaluation studies reported in Tables 2and 3 were performed with 2B8 derived from, a CHO cell expressionsystem; The MX-DTPA conjugate was using a protocol similar to that usedfor the previously characterized 2B8-49. Reactions were performed usingapproximately 3 mg of antibody and a 4:1 molar ratio of chelator toantibody as described in co-owned, copending application Ser. No.09/              , concurrently filed and herein incorporated byreference.

Immunoreactivities for Y2B8 preparations were determined using themethod of Lindmo et al. Increasing amounts of freshly harvested CD20positive SB cells were incubated with a fixed amount of Y2B8 underconditions of antigen excess. Reciprocal plot analysis of the bindingdata showed an immunoreactivity of 72.2% for Y2B8 following one trialpreparation (FIG. 1).

Example 3 Evaluation of Further Reaction Parameters

I. Introduction

Experiments described in this section examine the impact of protocoldeviations on the binding of Y2B8 prepared using the Y2B8 RadiolabelingKit. Binding of a radiolabeled antibody may be affected by severalparameters during the radiolabeling process (Table 4).

TABLE 4 Predicted Predicted Effect on Effect on Radiolabeling KitDeviation Labeling Conditions Binding 1.) Adding Excess Volume of ⁹⁰Ydecrease pH; decrease increase radiolysis 2.) Adding Less Volume of ⁹⁰Yno change in pH; increase decrease radiolysis or none 3.) Adding ExcessVolume of NaAc no change in pH; increase decrease radiolysis or none 4.)Adding Less Volume of NaAc decrease pH; decrease increase radiolysis 5.)Adding Excess Volume of no change in pH; increase 2B8-MX-DTPA decreaseradiolysis (lower specific activity) 6.) Adding Less Volume of no changein pH; decrease 2B8-MX-DTPA increase radiolysis (higher specificactivity) 7.) Incubating >5 min. increase radiolysis decrease 8.)Incubating <5 min. decrease radiolysis increase or none

The following deviations from the radiolabeling protocol were identifiedas those most likely to have a negative impact on binding, and included:1.) addition of a lesser volume of sodium acetate 2.) addition of excess⁹⁰Y chloride solution 3.) addition of a lesser volume of 2B8-MX DTPA and4.) exceeding the maximum reaction incubation time. The impact of thesedeviations was evaluated separately and simultaneously.

When evaluated separately, 20% volume deviations in items 1-3 aboveresulted in IDEC-Y2B8 passing the release specification established forbinding in the clinical trial, even when incubated for 8 min. In a studywhere all three volume deviations (1-3 above) were made simultaneously,only doses prepared using a Monday labeling protocol potentially themost radiolytic) and incubated for 8 min. (60% longer than normal) weremarginally below (<3%) the clinical release specification. In contrast,doses prepared using a Friday labeling protocol maintained acceptablebinding results, despite the cumulative effects of deviations in allfour parameters (1-4 above). For all deviations, separately andcollectively, radioincorporation was above the clinical releasespecification of 95%.

II. Choice of Parameters

We decided that a 20% deviation from the required reagent volumes, orallowing the reaction time to exceed by 30% the maximum 6 min. usednormally, represented potentially extreme deviations from the protocolused in the radiopharmacy. In this study we evaluate the impact of thesedeviations on the binding of IDEC-Y2B8. We simulated “Monday” and“Friday” labelings to insure that the conditions evaluated representedextremes of dose preparation for the entire week. We also evaluated thecombined effect on binding when all deviations occur in a single dosepreparation, and the impact of these deviations on radioincorporation ofthe ⁹⁰Y.

“Monday” and “Friday” labelings are a reflection of the concept that,since the ⁹⁰Y chloride solution has a short half-life (64 hr), thevolume of the radioisotope used depends on the day of the week a dose isprepared. For this reason, the reaction volume for a dose prepared on aMonday is smaller, resulting in higher a ⁹⁰Y concentration, possiblyresulting in greater radiolysis. Therefore, we simulated Monday andFriday labeling procedures to insure that the conditions evaluatedrepresented extremes of dose preparation for the entire week.

III. Materials and Methods

A. Reagents

-   1. ⁹⁰YCl₃ in 0.05 M HCL; Pacific Northwest National Laboratory,    reagent grade; P.O.#08016, 08118-   2. Ultrex HCL; J. T. Baker, Product#6900, Lot# J22539-   3. Sterile Water for Irrigation; Baxter, Part#2F7114, Lot# G924092-   4. Dilution Buffer; contains 10 mM phosphate buffered saline, pH    7.4, 1% BSA; Sigma, Part# P-3688, Lot#076H8913-   5. IDEC Supplied Radiolabeling Kit; IDEC Part#130018, Lot#0129,    containing the following:    -   a.) 2B8-MX-DTPA; IDEC Part#129017, Lot#0165    -   b.) 50 mM Sodium Acetate; IDEC Part#121017, Lot#0209A    -   c.) Formulation Buffer; IDEC Part#120015, Lot#0202    -   d.) Reaction Vial; IDEC Part#122015, Lot#0218-   6. Lyophilized SB Cells, IDEC Part#127, Lot#127-001F    B Materials and Equipment-   1. Pipettors (20, 200 and 1000 gL)-   2. Vortexer-   3. Metal-Free Pipette Tips (Biorad; metal-free)-   4. Gamma Counter (Isodata, Model#20-10)-   5. Glass Tubes (12×75 mm)-   6. Polypropylene Tubes (Costar; 15 mL and 50 mL conical, sterile)-   7. Tec-Control Radiochromatographic Kit (Biodex; Cat#151-770)-   8. Microcentrifuge (Savant)-   9. Polypropylene microfuge tubes, metal-free (Biorad; Cat#223-9480)    C. Methods    1. Preparation of Y2B8

In general, ⁹⁰Y-labeled 2B8-MX-DTPA was prepared using a small-scaleversion of the radiolabeling kit protocol described above as modified bythe changes described below. Radiolabeling was performed using ⁹⁰Ychloride stock concentrations of 84 mCi/mL or 29.8 mCi/mL to simulate,respectively, Monday or Friday dose preparations (based on a Wednesdaycalibration of 50 mCi/mL). The concentrated ⁹⁰Y chloride solution wasdiluted using 50 mM HCl (Ultrex, high-purity) in plastic “metal-free”microfuge tubes. The Ultrex (high-purity) HCl was diluted to 50 mM withSterile Water for Irrigation (SWFI). Radiolabeling reactions wereperformed in plastic “metalfree” microfuge tubes, 15 mL conical tubes,or 10 mL glass septum Reaction Vial provided in the Y2B8 RadiolabelingKit.

a. Small-Scale Labeling to Predict Full-Scale Dose Preparations

Radiolabeling reactions of 1, 3, 10, and 40 mCi were performed usingreaction conditions simulating a Monday dose preparation. Reagentvolumes in mLs for each reaction are summarized in Table 5.

TABLE 5 Volume of Reagents (mL) ⁹⁰Y Amount (mCi) ⁹⁰Y Chloride SodiumAcetate 2B8-MX-DTPA 1 0.0119 0.0143 0.0333 3 0.0357 0.0429 0.0998 100.119 0.143 0.333 40 0.476 0.571 1.33

After a 5 min. incubation, 20 μL samples were removed and diluted withFormulation buffer to a final antibody concentration of 0.21 mg/mL andstored at 2-8° C. until assayed. Binding values were normalized to the 1mCi reaction because 1 mCi reactions were used as controls in allsubsequent experiments described in this report. Values reported werenormalized to the 1 mCi control sample by dividing the binding value foreach reaction by the binding value for the control, expressed as apercentage.

b. Impact of Adding Volume of Sodium Acetate

For a Monday labeling, 10 mCi of ⁹⁰Y chloride (0.119 mL) was mixed with0.114 mL of 50 mM sodium acetate. This volume of 50 mM sodium acetaterepresents a 20% decrease in the amount of buffer normally used toprepare clinical doses of IDEC-Y2B8. Conjugated antibody (2B8-MX-DTPA)was added (0.333 mL), the sample mixed and then incubated at ambienttemperature. Specific activity of the radiolabeling solution was 18.9mCi/mg antibody. At 2 min., 0.020 mL was removed, formulated to 0.24mg/mL with formulation buffer, and stored at 2-8° C. The remainder ofthe radiolabeling solution was formulated, after 8 min., to 0.24 mg/mLand stored at 2-8° C. The protocol was repeated to simulate a Fridaylabeling, using 0.336 mL of ⁹⁰Y chloride, 0.323 mL of sodium acetate,and 0.333 mL of 2B8-MX-DTPA. For both studies, the 1 mCi controlreaction was performed using the “standard” conditions described above(5 min. reaction).

c. Impact of Adding Excess Volume of ⁹⁰Y Chloride

For a Monday labeling, 12 mCi of ⁹⁰Y chloride (0.143 mL) mixed with0.143 mL of 50 mM sodium acetate. This volume of ⁹⁰Y represents a 20%increase in the amount of ⁹⁰Y required for a typical dose preparation ofY2B8. Conjugated antibody was added and the sample solution mixed andincubated at ambient temperature. The final specific activity was 22.5mCi/mg antibody. At 2 min., 0.020 mL was removed, formulated to 0.24mg/mL with formulation buffer, and stored at 2-8° C. After 8 min., theremainder of the radiolabeling solution was formulated to 0.24 mg/mL andstored at 2-8° C. Friday labeling was performed similarly using 0.403 mLof ⁹⁰Y chloride, 0.403 mL of 50 mM sodium acetate, and 0.333 mL of2B8-MX-DTPA (specific activity 22.5 mCi/mg antibody). For both studies,a 1 mCi control reaction was performed using the “standard” conditionsdescribed above (5 min. reaction).

d. Impact of Adding Less Volume of Antibody Conjugate

For a Monday labeling, 10 mCi of ⁹⁰Y chloride (0.119 mL) was mixed with0.143 mL 50 mM sodium acetate. Conjugated antibody (0.267 mL) was added,representing 20% less antibody than normally used, the solution mixedand incubated at ambient temperature. At 2 and 8 min., 0.020 mL wasremoved, formulated with Formulation buffer to a final antibodyconcentration of 0.21 mg/mL, and stored at 2-8° C. until assayed. AFriday labeling was performed similarly using 0.336 mL ⁹⁰Y chloride,0.403 mL of 50 mM sodium acetate, and 0.27 mL conjugate. For bothstudies, a 1 mCi control reaction was performed using the “standard”conditions described above (5 min. reaction).

e. Impact of Combined Reagent Deviations

The impact of a 20% deviation in volume of sodium acetate, ⁹⁰Y chloride,and conjugate was assessed simultaneously for a Monday or a Fridaylabeling protocol. For a Monday labeling, 12 mCi of ⁹⁰Y (0.143 mL) wasmixed with 0.114 mL 50 mM sodium acetate, representing a 20% increase inthe amount of ⁹⁰Y chloride and a 20% decrease in the amount of sodiumacetate normally used. 2B8-MX-DTPA (0.267 mL), representing 20% lessantibody than normally used, was added and the reaction mixtureincubated at ambient temperature. At 2, 4, 6, and 8 min., 0.020 mL wasremoved from the reaction mixture, formulated with Formulation buffer toa final antibody concentration of 0.21 mg/mL, and stored at 2-8° C.until assayed. Friday labeling was performed similarly using 0.403 mL⁹⁰Y chloride, 0.387 mL of sodium acetate, and 0.267 mL of conjugate; 40μL samples were removed at the indicated times and formulated withFormulation buffer. For both studies, a 1 mCi control reaction wasperformed using the “standard” conditions described above (5 min.reaction).

2. Determination of Radioincorporation

The amount of radioactivity associated with the conjugates wasdetermined according to the assay described above, using thecommercially available kit manufactured by Biodex (Tec-ControlRadiochromatographic Kit). In general, 0.5-1 μL samples were applied toduplicate strips using a micropipetter and developed according to theBiodex instructional insert. Strip halves were counted for radioactivityin glass tubes using an Isodata gamma counter with window to 100-1000KeV. The radiolabel incorporation was calculated by dividing the amountof radioactivity in the top half of the strip by the total radioactivityfound in both top and bottom halves. This value was expressed as apercentage and the mean value determined.

3. Determination of Binding

Samples were analyzed for percent binding to CD20 positive cellsfollowing the protocol described above. However, the negative controlHSB Cell samples were not included in these experiments, and the SBCells were lyophilized in 5 mL vials instead of microfuge tubes.

Essentially, all final formulated Y2B8 samples were diluted 1:100 withDilution buffer (10.0 μL antibody+990 [μL buffer). The antibody wassubsequently diluted again to an approximate concentration ranging of 8ng/mL by adding 35 μL of the 1:100 dilution to 10 mL of dilution bufferin a 50 mL polypropylene tube.

Six to seven vials of lyophilized cells were reconstituted with SWFI andpooled in a 50 mL conical tube. Reconstituted cells (0.5 mL) were thenaliquoted out in triplicate into three 1.5 mL microfuge tubes, threetubes per sample being tested. Dilution buffer (0.5 mL) was added tothree empty microfuge tubes. Diluted antibody (0.5 mL) was added to eachtube, capped tightly, and incubated at ambient temperature for 45 min.with end-over-end mixing. After incubation, cells were pelleted bycentrifugation for 5 min. at a setting of “6” (4000×g) using a Savantmicrocentrifuge. Supernatants from the samples (0.75 mL) weretransferred to 12×75 mm glass tubes for radioactivity counting using anIsodata gamma counter with energy window settings of 100-1000 KeV.

Radioactivity bound (B) to cells was calculated by subtracting theunbound radioactivity (supernatant) from the total radioactivity added.Total radioactivity was determined from the radioactivity counted in thetubes without cells. Percent binding was calculated by expressing thebound radioactivity as a percentage of the total.

To minimize the effect of lot-to-lot variability of lyophilized cellsused to assess binding, binding values were normalized to 1 mCi Y2B8controls prepared using “standard” labeling conditions. Control sampleswere prepared, as stated earlier in this section, for each set ofexperiments.

D. Results

1. Small-Scale Labeling to Predict Full-Scale Dose Preparations

To insure that small-scale radiolabeling reactions were predictive offull-scale (40 mCi) dose preparations, 1, 3, 10, and 40 mCi Y2B8 doseswere prepared using the radiolabeling protocols described above. Theseresults are shown in Table 6 and demonstrate that increasing the scaleof the reaction mixture from 1 mCi to 40 mCi dose not adversely affectbinding or radioincorporation.

TABLE 6 Amount of ⁹⁰Y mCi % of Control Binding % Radioincorporation 1100 99.2 3 102 99.1 10 98.6 99.0 40 98.2 99.02. Impact of Adding Less Volume of Sodium Acetate

When Y2B8 was prepared using 20% less vole of 50 mM sodium acetate, andextending the incubation time by 60%, substantial binding was retained,compared to the radiolabeled antibody prepared following “standard”labeling conditions (Table 7 below). Even when the labeling reaction wasperformed using Monday labeling conditions, >89% of the control bindingwas retained. Similar results were obtained for a Friday dosepreparation. These deviations did not impact radioincorporation,regardless of the day the dose was prepared.

3. Impact of Adding Excess Volume of ⁹⁰Yttrium Chloride

When Y2B8 was prepared using a 20% excess volume of ⁹⁰Y chloride, incombination with an incubation time 60% longer than that used normally,substantial binding was retained, when compared to the control preparedaccording to “standard” labeling conditions (Table 7 below). When thelabeling reaction was increased to 8 min., binding still remained >90%,relative to the control, for either a Monday or a Friday dosepreparation. Adding 20% more volume of ⁹⁰Y chloride did not impactradioincorporation, regardless of the day the dose was prepared.

4. Impact of Adding Less Volume of Antibody Conjugate

When Y2B8 was prepared using a 20% less volume of the conjugate(2B8-MX-DTPA), and extending the incubation time by 60%, binding was notsignificantly affected, compared to Y2B8 prepared according to“standard” labeling conditions (Table 7 below). Adding 20% less volumeof conjugate did not impact radioincorporation, regardless of the daythe dose was prepared.

TABLE 7 Monday Dose Preparation^(a) Friday Dose Preparation^(b) % ofBinding % % of Binding % Labeling Deviation Control^(b)Radioincorporation Control^(b) Radioincorporation 1.) 20% Less 89.4 99.192.5 98.7 Volume of Sodium Acetate 2.) 60% Increase in Reaction Time (8min.) 1.) 20% Excess 90.6 99.1 91.8 98.6 Volume of ⁹⁰Y 2.) 60% Increasein Reaction Time (8 min.) 1.) 20% Less 98.9 99.0 98.7 98.6 Volume ofAntibody 2.) 60% Increase in Reaction Time (8 min.) ^(a)For a Mondaydose preparation, the concentration of ⁹⁰Y in the reaction solution is17 mCi/mL; the ⁹⁰Y concentration for a Friday labeling is 8 mCi/mL.Binding values normalized to labeled antibody prepared according toclinical dose protocol (RSBR-005) using “standard” reagent volumes and a5 min. reaction time.5. Impact of Combined Reagent Deviations

When Y2B8 was prepared using a protocol in which all four deviationswere made simultaneously, binding was still substantially maintained,compared to the radiolabeled antibody prepared using “standard” labelingconditions (Table 8 below). Binding was still >83%, even when a Mondaypreparation was incubated for 30% longer than the maximum 6 min. usednormally. Radioincorporation was not affected significantly by thesecumulative deviations, even after an 8 min. incubation time, regardlessof the day the dose was prepared.

TABLE 8 Labeling Time (min.) % of Binding Control % RadioincorporationMonday Dose Preparation 2 97.6 98.7 4 93.7 98.8 6 89.5 98.8 8 83.2 98.6Friday Dose Preparation 2 98.6 99.0 4 98.5 99.2 6 96.0 99.1 8 92.1 99.1V. Discussion

To reduce radiation exposure to operators, smaller labeling reactionswere evaluated instead of full-scale dose preparations. Therefore, weverified that the 1 mCi and 10 mCi labelings, evaluated in this study,were predictive of full-scale 40 mCi preparations. Results demonstratedno significant differences in binding and radioincorporation over arange of 1 mCi to 40 mCi.

We decided that 20% volume errors for sodium acetate, ⁹⁰Y chloride, andconjugated antibody represented potentially extreme deviations in theradiolabeling protocol. Additionally incubating for 8 min. (3 min.longer than normal) was viewed as a significant protocol deviation. Ingeneral, due to the short half life of ⁹⁰Y chloride, the volume ofradioisotope will differ depending on the day of the dose preparation.Therefore, experiments described in this report were performed using ⁹⁰Ychloride at concentrations representative of both Monday and Friday torepresent the full range of possible dose preparation.

Y2B8 doses prepared using 20% lesser volume of sodium acetate, andincubated for 8 min. retained significant binding (>89%) relative to thestandard labeling conditions. Similar results were obtained for dosesprepared Monday or Friday. This deviation in sodium acetate volume didnot affect radioincorporation.

Adding 20% more volume of ⁹⁰Y chloride, and incubating for up to 8 min.,reduced binding, relative to standard dose preparation conditions, forboth Monday and Friday. However, binding was still >90%, which is abovethe normalized release specification. Binding was marginally better fora Friday dose preparation. Radioincorporation was not significantlyaffected by the increased volume of ⁹⁰Y chloride.

To evaluate the impact of making simultaneously all volume deviations,Monday and Friday doses were prepared comparing 2, 4, 6, and 8 min.incubation times. Only when Y2B8 was prepared on a Monday, using an 8min. incubation time, does the binding marginally fail to meet thenormalized specification (83.2% compared to the normalized releasespecification of 86.3%).

REFERENCES

Each of the following citations is herein incorporated by reference:

-   M. W. Brechbiel et al., “Synthesis of C-Functionalized    trans-Cyclohexyldiethylenetriaminepenta-acetic Acids for Labelling    of Monoclonal Antibodies with the Bismuth-212 α-Particle    Emitter”, J. Chem. Soc. Perkin Trans., pp. 1173-1178, 1992-   M. E. Izard et al., “An Improved Method for Labeling Monoclonal    Antibodies with Samarium-153: Use of the Bifunctional Chelate    2-(p-Isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic    Acid”, Bioconjugate Chem., 3(4):346-350, 1992.-   R. B. Huneke et al., “Effective α-Particle-mediated    Radioimmunotherapy of Murine Leukemia¹”, Cancer Research,    52:5818-5820, 1992-   O. A. Gansow et al., “Macrocyclic or Conventional Ligands? Selection    of Effective Chelators for ⁹⁰Y or ²¹²Bi Radioimmunotherapy”,    Chemistry Section, Radiation Oncology Branch, Metabolism Branch,    National Cancer Institute-   S. Mirzadeh^(1,2) et al., “The Chemical Fate of ²¹²Bi-DOTA Formed by    β⁻ Decay of ²¹²Pb(DOTA)²⁻*′**”, Radiochimica Acta 60:1-10, 1993

1. A method for radiolabeling a chelator-conjugated antibody or antibodyfragment with a therapeutic radioisotope for administration to a patientcomprising (i) mixing the chelator-conjugated antibody or antibodyfragment with a solution comprising the therapeutic radioisotope or saltthereof; and (ii) incubating the mixture for a time between about 2 and10 minutes under amiable conditions such that a radiolabeled antibody orantibody fragment is produced having sufficient radioincorporation,immunoreactivity of at least 50% and a specific activity of about 5mCi/mg to about 20 mCi/mg, without further purification of theradiolabeled antibody or antibody fragment from unincorporatedradioisotope.
 2. The method of claim 1, wherein said chelator is abifunctional chelator selected from the group consisting of MX-DTPA,phenyl-DTPA, benzyl-DTPA, CHX-DTPA, DOTA, and derivatives thereof. 3.The method of claim 1, wherein the antibody fragment is selected fromthe group consisting of Fab, F(ab′)₂, and Fv fragments.
 4. The method ofclaim 1, wherein step (ii) comprises incubating the mixture at atemperature in the range of from about 25° C. to about 43° C.
 5. Themethod of claim 1, wherein a level of radioincorporation of greater than95% is achieved.
 6. The method of claim 1, wherein said therapeuticradioisotope is 90Y.
 7. The method of claim 6, wherein the pH of themixture of chelator-conjugated antibody or antibody fragment andsolution comprising the therapeutic radioisotope or salt thereof that isproduced in step (i) is in the range of about 3 to about
 6. 8. Themethod of claim 7, wherein the pH is adjusted with a sodium acetatesolution.
 9. The method of claim 6, wherein the specific activity of theradiolabeled antibody or antibody fragment is 10 mCi/mg to about 20mCi/mg.
 10. The method of claim 6, wherein the radiolabeled antibody orantibody fragment produced in step (ii) has at least 70%immunoreactivity.
 11. The method of claim 6, wherein said 90Y-labeledantibody or antibody fragment having sufficient radioincorporation, anda specific activity of about 5 mCi/mg to about 20 mCi/mg, such that itmay be administered directly to the patient without further purificationof the radiolabeled antibody or antibody fragment from unincorporatedradioisotope, is produced by an incubation time of between about 2 andten minutes.
 12. The method of claim 11 wherein said 90Y-labeledantibody or antibody fragment is produced by an incubation time ofbetween about 2 and less than about eight minutes.
 13. The method ofclaim 6, wherein the antibody or antibody fragment binds specifically toCD20.
 14. The method of claim 13, wherein the antibody is 2B8.
 15. Themethod of claim 14, wherein the chelator is MX-DTPA.
 16. The method ofclaim 15, wherein a level of radioincorporation greater than 96% isachieved.
 17. The method of claim 1, further comprising diluting theradiolabeled antibody or antibody fragment to an appropriateconcentration in formulation buffer for administration to a humanpatient without further purification of the radiolabeled antibody orantibody fragment from unincorporated radiolabel.
 18. The method ofclaim 17, wherein the mixture is incubated for a time of about 2 toabout 5 minutes.
 19. The method of claim 1, further comprising dilutingthe radiolabeled antibody or antibody fragment to an appropriateconcentration in formulation buffer for administration to a humanpatient without further purification of the radiolabeled antibody orantibody fragment from unincorporated radiolabel.
 20. The method ofclaim 19, wherein the formulation buffer comprises a radioprotectant andan unconjugated chelator.
 21. The method of claim 20, wherein theradioprotectant is selected from the group consisting of human serumalbumin (HSA), ascorbate, ascorbic acid, and a free radical scavengerselected from the group consisting of phenol, sulfites, glutathione,cysteine, gentisic acid, nicotinic acid, ascorbyl palmitate, HOP(:O)H₂,glycerol, sodium formaldehyde sulfoxylate, Na₂S₂O₅, Na₂S₂O₃, and SO₂.22. The method of claim 20, wherein the unconjugated chelator is DTPA orEDTA.
 23. A method for radiolabeling a MX-DTPA-conjugated antibody orantibody fragment with ⁹⁰Y for administration to a patient comprising(i) mixing the MX-DTPA-conjugated antibody or MX-DTPA-conjugatedantibody fragment with a solution comprising ⁹⁰Y or salt thereof; and(ii) incubating the mixture for a time between about 2 and 10 minutesunder amiable conditions such that a ⁹⁰Y-labeled antibody or antibodyfragment is produced having sufficient radioincorporation,immunoreactivity of at least 50% and a specific activity of about 5mCi/mg to about 20 mCi/mg, without further purification of the⁹⁰Y-labeled antibody or antibody fragment from unincorporated ⁹⁰Y. 24.The method of claim 23, wherein a level of radioincorporation greaterthan 96% is achieved.
 25. The method of claim 23, wherein the antibodyor antibody fragment binds specifically to CD20.
 26. The method of claim23, further comprising diluting the 90Y-labeled antibody or antibodyfragment to an appropriate concentration in formulation buffer foradministration to a human patient without further purification of the90Y-labeled antibody or antibody fragment from unincorporated 90Y. 27.The method of claim 26, wherein the mixture is incubated for a time ofabout 2 to about 5 minutes.
 28. The method of claim 26, wherein theradioprotectant is selected from the group consisting of human serumalbumin (HSA), ascorbate, ascorbic acid, phenol, sulfites, glutathione,cysteine, gentisic acid, nicotinic acid, ascorbyl palmitate, HOP(:O)H₂,glycerol, sodium formaldehyde sulfoxylate, Na₂S₂0₅, Na₂S₂0₃ and SO₂. 29.The method of claim 28, wherein the formulation buffer further comprisesan unconjugated chelator selected from the group consisting of DTPA andEDTA.